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A Suite of Novel Human Spermatozoal RNAs

G. CHARLES OSTERMEIER^*,

ROBERT J. GOODRICH^*,

STEPHEN A. KRAWETZ^*,,

From the ^* Department of Obstetrics and Gynecology, Center for Molecular Medicine and Genetics, and Center for Scientific Computing, Wayne State University, Detroit, Michigan.

Correspondence to: Stephen A. Krawetz, Charlotte B. Failing Professor of Fetal Therapy and Diagnosis, 253 C.S. Mott Center, 275 East Hancock, Detroit, MI 48201 (e-mail: steve{at}compbio.med.wayne.edu).

Received for publication June 20, 2004; accepted for publication August 2, 2004.

	Abstract

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We recently described a complex population of spermatozoal coding RNAs that are delivered to the oocyte on fertilization. These are derived throughout spermatogenesis, representing a record of past events. Recently, evidence has been provided that micro-RNAs are present in testes, suggesting that they might also be carried in ejaculate spermatozoa. To directly test this hypothesis, a unique microarray system capable of directly identifying antisense RNAs and predicted transcripts was utilized. RNA isolated from the ejaculate spermatozoa of 6 normal fertile men was directly hybridized to sense oligonucleotide arrays containing 10 000 elements. This revealed 68 shared RNAs, some of which are similar to those previously defined as micro-RNAs, whereas others were the antisense of previously in silico-predicted transcripts. The results and implications of this study are described in this communication.

     Key words: Development, fertility, cloning, microarray, novel transcripts, antisense RNA, spermatozoa.

	Materials and Methods

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RNA Extraction

Six human ejaculates were collected by masturbation and allowed to liquefy for 30 minutes. The semen was washed 3 times in frozen storage buffer (50 mM HEPES buffer, pH 7.5; 10 mM NaCl; 5 mM Mg-acetate; and 25% glycerol), then stored at -80°C. Subsequent to storage, the samples were thawed, washed twice in 12 mL of PBS, then resuspended in a somatic cell lysis buffer (0.1% SDS, 0.5% Triton X-100 in dH₂O). This procedure produces essentially pure spermatozoa (Ostermeier et al, 2002b). Cell concentrations were determined with a hemacytometer. Ribonucleic acid was extracted from the purified spermatozoa with the RNeasy kit (Qiagen Inc, Valencia, Calif) with minor modifications. Lysis buffer was added to the samples at 600 µL/10⁷ cells. The lysates were homogenized with a 26-gauge needle and heated for 30 minutes at 65°C. After incubation, the samples were homogenized a final time to ensure shearing of the DNA. This sample was then loaded onto the RNeasy column and processed essentially as described by the manufacturer. The RNA was eluted from the column with 2 (50-µL) RNase-free H₂O washes. Dithiothreitol at a final concentration of 5 mM and 40 units of RNase Block (Strategene, La Jolla, Calif) were then added to the samples before a 20-minute treatment with RNase-free DNase I (Ambion, Austin, Tex) at 37°C. The amount of RNA was then determined with the RiboGreen RNA quantifying kit (Molecular Probes, Eugene, Ore) as previously described (Goodrich et al, 2003). The samples were stored at -80°C until use.

Quality Control

Sample purity was established by reverse transcription polymerase chain reaction (RT-PCR), employing the intron-spanning protamine 2 (PRM2) primers. Only a 149-bp intronless product should be observed. The presence of contaminating DNA, as evidenced by a 310-bp product, prompted an additional DNase I treatment. RT-PCR was performed with oligo-dT primers and the SuperScript III enzyme (Invitrogen Co, Carlsbad, Calif) and 100-200 ng of total RNA. PCRs were performed for 40 cycles with a cDNA copy of the purified RNA as template, along with the following intron-spanning PRM2 primers (forward: -tat agg cgc aga cac tgc; reverse -gcc ttc tgc atg ttc tct) and the Hot-Star Taq Polymerase system (Qiagen).

Labeling and Hybridization

Hybridizations were carried out with the Hybrid Capture HC Express Array Kit (Digene, Gaithersburg, Md). Briefly, 500 ng of isolated RNA was denatured at 95°C for 2 minutes in 25 µL of hybridization buffer. MWG sense oligonucleotide arrays (MWG Biotech, Ebersberg, Germany) under LifterSlips (Electron Microscopy Sciences, Washington, Pa) were then overlayed with the hybridization mix by capillary action. The hybridization chambers were sealed and then incubated overnight at 65°C. Following hybridization, the arrays were washed, allowed to air dry for 1 minute, then returned to the hybridization chambers. RNase (50 µL) containing primary RNA/DNA hybrid antibody was placed under the LifterSlips, and the sample was incubated for 60 minutes at room temperature. The microarrays were then washed and stained with 50 µL of the secondary antibody at room temperature. Samples were then washed a final time before signal enhancement, as described by the manufacturer. The microarrays were dried then scanned with a Typhoon 9210 scanner (Amersham Pharmacia Biotech, Piscataway, NJ).

Analysis

The scanned images were viewed with Quantity One software (BioRad, Hercules, Calif) and saved in the TIFF format. The saved images were analyzed with the Imagene software (Bio-Discovery, El Segundo, Calif) with default settings for threshold (ie, 2 SD above background) and spot detection. A list of positive hybridizations was generated for each microarray and hybridizations were compared with Statistical Analysis Software (SAS, Cary, NC) as described by Ostermeier et al (2002a).

	Results and Discussion

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To examine whether "noncoding" strand RNAs were present in human spermatozoa, the MWG 30k (A, B, and C) 50-mer sense oligonucleotide arrays (MWG Biotech) were directly probed with RNA isolated from ejaculate spermatozoa. All RNA-DNA oligonucleotide hybrids were detected with a fluorescently tagged RNA-DNA antibody (Digene). To maximize specificity, stringent hybridization temperatures and washes were employed. The calculated melting temperature (Howley et al, 1979) of each arrayed 50-mer element indicated that a perfectly matched 20-mer could yield a hybridization signal. Only the C-array that predominantly contained in silico-identified genes showed significant hybridization. Specificity was further confirmed because the 104 Arabidopsis controls present on each chip did not hybridize to the added RNA probe. It was thus selected for further analysis of spermatozoal RNAs from 6 different men. Each of the ejaculate profiles from the C-chip was then compared. A core set of 68 candidate spermatozoal sense-hybridizing RNAs shared among all 6 individuals was identified, as summarized in Table 1. The 68 core sequences represented by each 50-mer were compared with the mature segment of the miRNAs of the human genome (Lim et al, 2003). Eighteen of the 50-mers corresponding to the candidate spermatozoal RNAs that hybridized to the sense array had an alignment z-score of greater than 100 (http://compbio.med.wayne.edu/Sperm_RNAi_seq.htm). The alignment of one of these miRNAs contained in the human miRNA registry of complete miRNA sequences (http://www.sanger.ac.uk/Software/Rfam/mirna/index.shtml) could be extended throughout its corresponding 50-mer. This sequence aligned with mir-182. Given that at least 15% of the miRNAs remain to be identified (Lim et al, 2003), these data support the notion that novel spermatozoal noncoding antisense RNAs are present.

To begin to define their role, the potential targets of these 68 shared sequences were mined with the ENSEMBL (http://www.ensembl.org/) and SOURCE databases (http://genome-www5.stanford.edu/cgi-bin/source/sourceSearch) to identify cytogenetic location and overlap with known transcripts, as well as their tissue and functional distributions in early development. As summarized in Table 2, the corresponding transcripts that have been associated with early development were evenly distributed within the genome and appeared to be expressed in a wide variety of tissues, including the testes and early embryonic tissues. These include DKK2, TIA, and FAT3. For example, dickkopf2 (DKK2) has been shown to inhibit the wingless type (WNT) signaling pathway (Mao et al, 2001; Mao and Niehrs, 2003) that has been implicated in oncogenesis and in several developmental processes, including the regulation of cell fate and pattern formation during embryogenesis. Thus, silencing of DKK2 with antisense RNAs delivered by the spermatozoa could ensure a functional WNT pathway.

The results presented in this study provide additional evidence for the existence of a wide spectrum of spermatozoal RNAs. On one hand, these exciting observations provide clear evidence that previously unknown or in silico-predicted transcripts can be identified by the application of microarrays to spermatozoal RNAs. This is interesting in light of the recent and fruitful efforts to test these various predictions (Rinn et al, 2003). These studies have indeed shown that our information content is larger than expected. On the other hand, as we have shown, this population houses a suite of noncoding strand RNAs that could include miRNAs. Consistent with our previous observations that spermatozoa contain testis transcripts (Ostermeier et al, 2002b), these are likely similar to the micro-RNAs recently identified in human testis (Liu et al, 2004). It is tempting to speculate that the delivery of these spermatozoal "antisense" RNAs on fertilization enables their participation in early postfertilization processes. They may provide a new level of control that helps to establish imprints during the transition from maternal to embryonic genome, or both. The latter could be the case given the recent observations of Morris et al (2004) and Fukagawa et al (2004), showing that this class of RNAs in mammals can confer transcriptional silencing by methylation, a known mechanism of imprinting. Their results and the findings reported in this communication should spur our understanding of the functions of spermatozoal RNAs.

	Acknowledgments

 
Normal fertile human sperm samples were a generous gift from the NICDH Reproductive Medicine Network (U10 HD-39005).

	Footnotes

 
This work was supported by WSU Ob/Gyn grant 95200 to S.A.K.

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